How obligatory is anaerobiosis?

Historically many bacteria have been classified as obligate anaerobes. They have been construed as wholly intolerant of oxygen, a feature that was originally ascribed to their lack of superoxide dismutases and catalases. Clostridial species were regarded as classic examples. We now know that this view is quite wrong: enzymes that scavenge superoxide, hydrogen peroxide and even oxygen itself abound in anaerobes. In the current issue of Molecular Microbiology, Hillmann et al. demonstrate that full production of these proteins can allow Clostridium acetobutylicum to survive and even grow in oxygenated culture medium. Evidently, oxidative defences in anaerobes can be robust. In all likelihood, they are critical for the movement of bacteria through aerobic environments to new anaerobic habitats.     

有机垃圾发酵过程中的微生物研究

采用最大概率法和平板计数法[1] 测定城市有机垃圾堆肥过程中主要微生物类群的变化。实验表明 :中、高温好氧细菌在前 7d呈上升趋势 ,在第 7d达其最大值为 1.32× 10 13 /g干垃圾 ;随后 ,其数量随着氧含量的降低而逐渐下降。厌氧菌在前 10d呈上升趋势 ,到第 10d达到最大值 7.5 1× 10 5/ g干垃圾 ;随后随着营养物质的减少而减少。停止通气后 ,好氧菌中的兼性厌氧菌逐渐增多 ,严格好氧菌逐渐减少 ;厌氧菌中的兼性厌氧菌逐渐减少 ,而严格厌氧菌逐渐增多。高温放线菌 ,中温好、厌氧纤维素菌和高温好氧纤维素菌在发酵的前 4d均呈上升趋势 ,随即逐渐下降。中温放线菌和真菌只在发酵之初存在 ,其数量随着温度的上升和氧含量的下降而迅速下降至零。高温厌氧纤维素菌的数量一直在慢慢上升     

A modified tube method for the cultivation and enumeration of anaerobic bacteria.

A new type of tube (the Lee tube) has been developed for use in the cultivation and enumeration of obligate anaerobes. The Lee tube is a double-walled, screw-capped tube which allows the formation of a thin cylinder of agar medium between the two walls. Anaerobiosis is achieved through deoxygenation of the deep cylinder of agar during sterilization, a minimum of head space, and use of a reducing agent to absorb oxygen introduced during the inoculation procedure. For several species of Clostridium, Bacteroides fragilis, Fusobacterium necrophorum, Veillonella alcalescens, and Pectinatus cerevisiiphilus, colony counts of cultures in the Lee tubes were comparable with those obtained in pour plates incubated in a BBL GasPak system and in anaerobic roll tubes.     

Amorphous ferrous sulfide as a reducing agent for culture of anaerobes.

Amorphous ferrous sulfide, prepared by reacting ferrous ammonium sulfate and sodium sulfide, is an excellent reducing agent for the culture of anaerobes. It reduces resazurin and reacts much more rapidly with O2 than does either soluble sulfide (HS)- or cysteine. One of the end products of the oxidation of ferrous sulfide with O2 is red and serves as an indicator for the oxygen contamination of a culture medium. Amorphous ferrous sulfide served as a suitable reducing agent for the growth of species of Methanobacterium or Clostridium. Its use is recommended for enrichment or culture of anaerobes (e.g. autotrophs, fermentative organisms) from sediments and other habitats were organic reducing agents are undesirable and where soluble sulfide might be toxic.     

Amorphous ferrous sulfide as a reducing agent for culture of anaerobes.

Amorphous ferrous sulfide, prepared by reacting ferrous ammonium sulfate and sodium sulfide, is an excellent reducing agent for the culture of anaerobes. It reduces resazurin and reacts much more rapidly with O2 than does either soluble sulfide (HS)- or cysteine. One of the end products of the oxidation of ferrous sulfide with O2 is red and serves as an indicator for the oxygen contamination of a culture medium. Amorphous ferrous sulfide served as a suitable reducing agent for the growth of species of Methanobacterium or Clostridium. Its use is recommended for enrichment or culture of anaerobes (e.g. autotrophs, fermentative organisms) from sediments and other habitats were organic reducing agents are undesirable and where soluble sulfide might be toxic.     

Use of the Etest method for antimicrobial susceptibility testing of obligate anaerobes]

The aim of this study was to evaluate Etest usefulness for antimicrobial susceptibility testing of obligate anaerobes and to compare the activity of five antibacterial drugs against clinical strains of anaerobes. One hundred strains of obligate anaerobes were tested: 2 reference strains (B. fragilis ATCC 25285 and B. thetaiotaomicron ATCC 29741) and 98 clinical strains isolated from patients of the Infant Jesus Clinical Hospital--Center for Trauma Treatment in Warsaw during the last three years (1997-1999). Strains of seven genera of obligate nonsporeforming anaerobes (Bacteroides, Prevotella, Porphyromonas, Fusobacterium, Peptostreptococcus, Propionibacterium and Actinomyces) and strains of two sporeforming species (C. perfringens and C. difficile) were examined. The MIC values were determined by the gradient diffusion method Etest (AB BIODISK, Sweden). Wilkins-Chalgren solid medium supplemented with 5% of sheep blood was used. Test plates were incubated at 35 degrees C for 48 hours in glove-box (85% N2, 10% H2, 5% CO2). The MIC values for each strain and antimicrobial agent, and the MIC ranges for bacteria of the same species were established. Ten strains resistant to clindamycin, ten resistant to piperacillin, and ten resistant to imipenem were detected. Seven strains were resistant to metronidazole and two strains to piperacillin combined with tazobactam. Tazobactam restored the susceptibility of eight strains to piperacillin. Obtained results confirm that Etest method is useful for antimicrobial susceptibility testing of obligate anaerobes. Older (clindamycin and metronidazole) and newer (piperacillin, piperacillin/tazobactam and imipenem) antimicrobial agents revealed high and comparable activity against clinical strains of obligate anaerobes. The percentage of strains susceptible to tested antimicrobials was > or = 90. These antimicrobials may be still useful in the empiric treatment of infections caused by medically important anaerobes.     

人源溶菌酶基因LYZL4在毕赤酵母中的重组表达及活性测定

LYZL4是本实验室克隆的一种c型人溶菌酶基因,根据毕赤酵母密码子偏爱性对LYZL4基因进行优化设计,优化后的基因克隆至具有乙醇氧化酶启动子(AOX1)的pPIC9K载体中。通过电转化方法整合到甲醇营养型毕赤酵母表达菌株GS115基因组上,再利用不同浓度梯度的G418抗性YPD固体培养基筛选高拷贝转化子,经摇瓶发酵后,其上清液在SDS-PAGE胶上14.4 kDa处出现明显的蛋白条带,该蛋白条带经质谱鉴定证实是人LYZL4蛋白。以人溶菌酶(164 071U/mg)作为标准品,使用艳红微球菌作为底物,通过比色法测定重组LYZL4溶菌酶蛋白的活性,结果显示重组人溶菌酶LYZL4蛋白具有明显的溶菌活性,其酶活力可达38893U/ml。     

Dysbiosis in inflammatory bowel diseases: the oxygen hypothesis

The healthy intestine is characterized by a low level of oxygen and by the presence of large bacterial communities of obligate anaerobes. Dysbiosis of the gut microbiota has been reported in patients suffering from inflammatory bowel diseases (IBDs), but the mechanisms causing this imbalance remain unknown. Observations have included a decrease in obligate anaerobes of the phylum Firmicutes and an increase in facultative anaerobes, including members of the family Enterobacteriaceae. The shift of bacterial communities from obligate to facultative anaerobes strongly suggests a disruption in anaerobiosis and points to a role for oxygen in intestinal dysbiosis. Proposals to evaluate this hypothesis of a role for oxygen in IBD dysbiosis are provided. If this hypothesis is confirmed, decreasing oxygen in the intestine could open novel means to rebalance the microbiota and could provide novel preventative or therapeutic strategies for IBD patients in whom current treatments are ineffective.     

Chromatin structure and induction-dependent conformational changes of human interferon-beta genes in a mouse host cell

Multiple copies of a human interferon-beta gene introduced into a mouse host cell line can be activated by induction with double-stranded RNA. Several induction-dependent changes of the chromatin structure could be traced by mapping techniques using four different agents [DNase I, micrococcus nuclease, bromoacetaldehyde and methidiumpropyl-EDTA X iron(II)]. Our data show that all copies of the interferon gene have adopted a very similar conformation in the host cell and respond to the inducing stimulus in a highly synchronous fashion. Detailed induction-specific changes were observed best with the chemical reagents which disclose a specific hypersensitive site within a sequence that has been shown to be required for the induction process (around position -80) and three other regions which, in addition to the transcribed region itself, gain single-strand character by an auxiliary process which can be mimicked by the addition of butyrate to the medium and may therefore involve histone hyperacetylation. Six discrete 'phased' nucleosomes are present upstream from the gene and are modulated by induction. At least four nucleosomes are located downstream. The interferon genes are largely protected from micrococcus nuclease in the inactive state. Gene activation increases access to micrococcus nuclease and DNase I indicating gross conformational changes on a higher level of chromatin structure.     

Aerotolerance of strictly anaerobic microorganisms and factors of defense against oxidative stress: a review

Effects of aerobic conditions on strictly anaerobic microorganisms belonging to diverse taxa (clostridia, acetogenic bacteria, lactic acid bacteria, bacteroids, sulfate-reducing bacteria, and methanogenic archaea) and differing considerably in their oxygen resistance have been reviewed, with emphasis on the role of aerotolerance in the ecology of anaerobes. Consideration is given to components of nutritive media for anaerobe culturing, which decrease the toxic effects of oxygen and there by contribute significantly to maintenance and storage of industrial cultures of strictly anaerobic microorganisms. Physiological and biochemical factors are described, accounting for the relative resistance of many strict anaerobes to oxygen and products of incomplete reduction thereof. Specific attention is given to regulation of enzymes of antioxidative defense, operating in the cells of strict anaerobes under the conditions of oxidative stress caused by oxygen, superoxide anion, or hydrogen peroxide.     

Aerotolerance of strictly anaerobic microorganisms and factors of defense against oxidative stress: A review

Effects of aerobic conditions on strictly anaerobic microorganisms belonging to diverse taxa (clostridia, acetogenic bacteria, lactic acid bacteria, bacteroids, sulfate-reducing bacteria, and methanogenic archaea) and differing considerably in their oxygen resistance have been reviewed, with emphasis on the role of aerotolerance in the ecology of anaerobes. Consideration is given to components of nutritive media for anaerobe culturing, which decrease the toxic effects of oxygen and there by contribute significantly to maintenance and storage of industrial cultures of strictly anaerobic microorganisms. Physiological and biochemical factors are described, accounting for the relative resistance of many strict anaerobes to oxygen and products of incomplete reduction thereof. Specific attention is given to regulation of enzymes of antioxidative defense, operating in the cells of strict anaerobes under the conditions of oxidative stress caused by oxygen, superoxide anion, or hydrogen peroxide.     

Hydrogen online monitoring based on thermal conductivity for anaerobic microorganisms

H₂-producing microorganisms are a promising source of sustainable biohydrogen. However, most H₂-producing microorganisms are anaerobes, which are difficult to cultivate and characterize. While several methods for measuring H₂ exist, common H₂ sensors often require oxygen, making them unsuitable for anaerobic processes. Other sensors can often not be operated at high gas humidity. Thus, we applied thermal conductivity (TC) sensors and developed a parallelized, online H₂ monitoring for time-efficient characterization of H₂ production by anaerobes. Since TC sensors are nonspecific for H₂, the cross-sensitivity of the sensors was evaluated regarding temperature, gas humidity, and CO₂ concentrations. The systems' measurement range was validated with two anaerobes: a high H₂-producer (Clostridium pasteurianum) and a low H₂-producer (Phocaeicola vulgatus). Online monitoring of H₂ production in shake flask cultivations was demonstrated, and H₂ transfer rates were derived. Combined with online CO₂ and pressure measurements, molar gas balances of the cultivations were closed, and an anaerobic respiration quotient was calculated. Thus, insight into the effect of medium components and inhibitory cultivation conditions on H₂ production with the model anaerobes was gained. The presented online H₂ monitoring method can accelerate the characterization of anaerobes for biohydrogen production and reveal metabolic changes without expensive equipment and offline analysis.     

Development of an apparatus for cultivation of anaerobic microorganisms

Summary A novel apparatus with an L-shaped test tube was developed for anaerobic cell cultivation. Anaerobic condition was achieved without the rigorous gassing with CO₂ during the various stages of medium preparation. dispensation and cell inoculation. The growth of both moderate and strict anaerobes in this apparatus were similar to those obtained with the glove box method.     

Silica gel media for isolating and studying bacteria under hydrostatic pressure.

Individual colonies of micrococcus euryhalis and of a marine bacterial isolate were grown in pour tubes under hydrostatic pressure. The medium was prepared in a silica sol, and gelation was effected at 4 degrees C by addition of salts to achieve concentrations found in seawater.     

Silica gel media for isolating and studying bacteria under hydrostatic pressure.

Individual colonies of micrococcus euryhalis and of a marine bacterial isolate were grown in pour tubes under hydrostatic pressure. The medium was prepared in a silica sol, and gelation was effected at 4 degrees C by addition of salts to achieve concentrations found in seawater.     

Mitochondrial DNA sequences reveal extensive cryptic diversity within a western American springsnail

We analysed cytochrome c oxidase subunit I and NADH dehydrogenase subunit I sequence variation among 29 populations of a widely ranging southwestern springsnail (Pyrgulopsis micrococcus) and 18 regional congeners. Cladistic analyses of these sequences depict P. micrococcus as a polyphyletic composite of five well-supported clades. Sequence divergences among these clades and subclades imply the possible occurrence of as many as seven or eight cryptic species in addition to P. micrococcus. Our finding that P. micrococcus contains multiple, genetically distinct and geographically restricted lineages suggests that diversification within this highly speciose aquatic genus has been structured in large part by the operation of terrestrial barriers to gene flow. However, these sequence data also indicate that recent dispersal among hydrographically separated areas has occurred within one of these lineages, which we attribute to passive transport on migratory waterbirds.     

Microplates with integrated oxygen sensing for medium optimization in animal cell culture

A new approach using microtiter plate cultivation with on-line measurement of dissolved oxygen (DO) was applied for medium optimization of mammalian cell culture. Applying dynamic liquid phase balance, oxygen uptake rates were calculated from the DO level and used as an indicator for culture viability. The developed method was successfully applied to optimization of the concentration of glucose, glutamine and inorganic salts for cultivation of a Chinese Hamster Ovary (CHO) cell line. Using 2³ full factorial central composite design, the optimum medium composition could be identified in one single run. The concentration of inorganic salts had a significant influence on cultivation. The developed method exhibits high potential to improve procedures of medium optimization for animal cell cultivation by allowing the investigation of large sets of potentially important variables in short time and with reduced effort.     

一次性厌氧菌培养平板的制作与应用

用一次性厌氧菌培养平板,对１０属１７种６７株厌氧菌和３０份临床标本进行培养,同时用厌氧手套箱及厌氧罐作对照培养,其培养效果与厌氧手套箱一致,优于厌氧罐,且用一次性厌氧菌培养平板进行培养具有操作简便,宜于推广应用等特点。     

Oxygen Sensitivity of Various Anaerobic Bacteria

Anaerobes differ in their sensitivity to oxygen, as two patterns were recognizable in the organisms included in this study. Strict anaerobes were species incapable of agar surface growth at pO(2) levels greater than 0.5%. Species that were found to be strict anaerobes were Treponema macrodentium, Treponema denticola, Treponema oralis n. sp., Clostridium haemolyticum, Selenomonas ruminatium, Butyrivibrio fibrisolvens, Succinivibrio dextrinosolvens, and Lachnospira multiparus. Moderate anaerobes would include those species capable of growth in the presence of oxygen levels as high as 2 to 8%. The moderate anaerobes could be exposed to room atmosphere for 60 to 90 min without appreciable loss of viability. Species considered as moderate anaerobes were Bacteroides fragilis, B. melaninogenicus, B. oralis, Fusobacteria nucleatum, Clostridium novyi type A, and Peptostreptococcus elsdenii. The recognition of at least two general types of anaerobes would seem to have practical import in regard to the primary isolation of anaerobes from source material.     

Fluorinert, an oxygen carrier, improves cell culture performance in deep square 96-well plates by facilitating oxygen transfer

In bioprocess development, the 96-well plate format has been widely used for high-throughput screening of production cell line or culture conditions. However, suspension cell cultures in conventional 96-well plates often fail to reach high cell density under normal agitation presumably due to constraints in oxygen transfer. Although more vigorous agitation can improve gas transfer in 96-well plate format, it often requires specialized instruments. In this report, we employed Fluorinert, a biologically inert perfluorocarbon, to improve oxygen transfer in 96-well plate and to enable the growth of a Chinese Hamster Ovary cell line expressing a recombinant monoclonal antibody. When different amounts of Fluorinert were added to the cell culture medium, a dose-dependent improvement in cell growth was observed in both conventional and deep square 96-well plates. When sufficient Fluorinert was present in the culture, the cell growth rate, the peak cell density, and recombinant protein production levels achieved in deep square 96-wells were comparable to cultures in ventilated shake flasks. Although Fluorinert is known to dissolve gases such as oxygen and CO(2), it does not dissolve nor extract medium components, such as glucose, lactate, or amino acids. We conclude that mixing Fluorinert with culture media is a suitable model for miniaturization of cell line development and process optimization. Proper cell growth and cellular productivity can be obtained with a standard shaker without the need for any additional aeration or vigorous agitation.